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Methods, compositions, and kits for generating nucleic acid products substantially free of template nucleic acid

a nucleic acid product and template technology, applied in the field of methods, compositions, kits, to achieve the effect of substantially free nucleic acid products, and reducing the difficulty of generating nucleic acid products

Inactive Publication Date: 2011-09-15
NUGEN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0005]Accordingly, the present invention provides improved methods for generation of ds DNA fragment molecules suitable for downstream applications including but not limited to large scale analysis.

Problems solved by technology

The limitations of current nucleic acid preparation techniques impact the ability to carry out large scale analysis of multiple parameters, as is required for, for example, the genotyping of multiple loci in the study of complex diseases, detecting the presence or absence of specific nucleic acid species in a sample, large scale sequencing and the like.

Method used

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  • Methods, compositions, and kits for generating nucleic acid products substantially free of template nucleic acid
  • Methods, compositions, and kits for generating nucleic acid products substantially free of template nucleic acid
  • Methods, compositions, and kits for generating nucleic acid products substantially free of template nucleic acid

Examples

Experimental program
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Effect test

example 1

Whole Transcriptome Amplification Followed by Random Priming to Generate dsDNA Products with Reduced (Degradation) of Input Amplified Products

[0200]Linear Whole Transcriptome Amplification is carried out using WT-Ovation Pico RNA Amplification kit (NuGEN Technologies Inc, San Carlos) following the manufacturer instructions (http: / / www.nugeninc.com / tasks / sites / nugen / assets / File / user_guides / userguide_wt_ov_pico.pdf) to generate template nucleic acid. The input total RNA for each reaction is 10 ng of total RNA samples (HeLa total RNA and Brain total RNA from Ambion and total RNA from a biological sample). The amplification is performed in the presence of all four dNTPs as well as dUTP to render the template nucleic acid susceptible to fragmentation by the combined action of UDG and DMED.

[0201]The amplified cDNA is purified using QIAquick® PCR Purification Kit, Cat. #28104 as describe in the User Guide (reference above).

[0202]Random priming and extension of the amplified cDNA (i.e. temp...

example 2

Preparation of Double Stranded Products of the Present Invention for High Throughput Sequencing

[0208]The double stranded amplification products of Example 1 are blunt ended using a single stranded exonuclease such as exo1 or exo7 for degrading single stranded overhangs. Alternatively a polymerase such as T4 DNA polymerase or Klenow DNA polymerase is utilized for filling in single stranded overhangs, or a combination of exonuclease and polymerase. The blunt ended double stranded products are then phosphorylated with T4 polynucleotide kinase. The phosphorylated double stranded nucleic acid amplification products are then ligated to adaptor nucleic acids. The amplification products are then encapsulated in a water-in-oil emulsion and amplified in a clonal fashion in the presence of particles comprising sequences complementary to the adaptor nucleic acids. The resulting particles comprising clonally amplified amplification products are then sequenced using a Genome Sequencer manufacture...

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Abstract

Methods, kits, and compositions are provided herein for the generation of double stranded DNA products suitable for downstream analysis.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 233,441, filed Aug. 12, 2009; U.S. Provisional Application No. 61 / 239,749, filed Sep. 3, 2009; and U.S. Provisional Application No. 61 / 242,706, filed Sep. 15, 2009, which applications are incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTION[0002]Large scale analysis of nucleic acids often requires amplification and further generation of products such as single stranded DNA (ss DNA) or double stranded DNA (ds DNA) that are either labeled, ready for labeling, or ready for ligation of desired adapters. These large scale analysis methods are evolving and comprise the use of various high or low density microarrays, high throughput sequencing, and other high throughput or highly parallel techniques. The limitations of current nucleic acid preparation techniques impact the ability to carry out large scale analysis of multiple parameters, as is required for, for exampl...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B50/06C12P19/34C12N9/24C12N9/12C12Q1/68
CPCC12P19/34C12Q1/6806C12Q1/6853C12Q2521/531C12Q2525/179C12Q2535/122C12Q2525/173C12Q2525/121C12Q2525/119C12Q2525/101
Inventor KURN, NURITHWANG, SHENGLONG
Owner NUGEN TECH
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